Understanding the molecular principles that regulate endomembrane organelle maintenance and biogenesis stands as a principle question in cell biology. Research to date has established the protein/membrane trafficking pathways that support endomembrane organelle maintenance and through these studies, the central importance of the ER as the site of resident endomembrane protein synthesis has been identified. The molecular basis for endomembrane organelle biogenesis remains, however, mysterious. The objective of the proposed research is to define the role of ER-directed mRNA localization in endomembrane organelle biogenesis. It is well established that the protein trafficking pathways of eukaryotic cells also direct the partitioning of the mRNA transcriptome between the two primary protein synthesis compartments in the cell, the cytoplasm and the endoplasmic reticulum (ER). Thus, mRNAs encoding secretory/integral membrane proteins are localized to the ER via the Signal Recognition Particle (SRP) pathway and mRNAs encoding cytosolic/nucleoplasmic proteins, which lack encoded signal sequences, retain default localization in the cytosol. Although this positive selection model is well established, we have recently reported that i) mRNAs encoding resident proteins of the endomembrane organelles (mRNAendo) can be localized to the ER via a SRP- and translation-independent pathway; ii) the mRNAendo cohort is bound to the ER via direct, ribosome-independent interactions; and iii) the mRNAendo cohort is distinguished by its exceptionally high ER enrichment. These findings suggest that mRNA localization serves an integral, though unexplored, role in organelle biogenesis. We hypothesize that the autonomous localization of endomembrane resident protein-encoding mRNAs to the ER, and their direct, ribosome-independent association with the ER membrane, represents a self-organization mechanism functioning in organelle biogenesis. This hypothesis will be tested in the following specific aims: 1) Establish, at a genome-wide scale, the subcellular partitioning patterns of endomembrane resident protein-encoding mRNAs; 2) Identify the molecular signals that direct endomembrane resident protein-encoding mRNAs to the ER; 3) Identify the RNA binding proteins and ER resident proteins that mediate ribosome-independent association of mRNAs with the ER membrane; 4) Determine if ribosome-independent binding of endomembrane protein encoding mRNAs to the ER is essential for organelle biogenesis. Genome-wide studies of mRNA partitioning in eukaryotic cells have revealed an unexpected complexity in the subcellular patterns of mRNA localization. In this proposal, we propose to extend recent discoveries of mRNA cohort-specific patterns of RNA localization to the ER to the question of organelle biogenesis. If successful, these studies will provide fundamental insights into the molecular principles of organelle biogenesis and support the development of therapeutic approaches to diseases of organelle dysfunction. PUBLIC HEALTH RELEVANCE: The organelles of eukaryotic cells perform numerous functions that are essential for cell viability. Indeed, numerous inherited metabolic diseases as well as diseases of ageing arise through disruptions in organelle function. The goal of this research is to identify the basic principles of how organelles are created and repaired, so that organelle-based diseases can be better understood and new therapies for organelle- based disease identified.